This application is based upon the hypothesis that intracellular antioxidant status is an important component of the endothelial cell dysfunction and characterizes atherosclerosis and aging and that contributes to the clinical manifestations of vascular disease. The endothelium normally maintains vascular homeostasis, in part, through the action of endothelium-derived nitric oxide (EDNO). Compelling evidence indicates that EDNO action is particularly sensitive to vascular oxidative stress (an imbalance between oxidants and antioxidants in favor of the former). Early research examining the mechanism(s) of impaired EDNO action involved antioxidant enzymes such as SOD or lipid-soluble only now being explored. The goal of this proposal is to define to role(s) of vitamin C and glutathione (GSH), the two principal intracellular water- soluble antioxidant species in vivo, on endothelial function. We will use human aortic endothelial cells (HAECs) as out experimental model and our index endothelial function will be the bioactivity of EDNO. In pursuing the goal of this project, we will consider the dual roles out of vitamin C and GSH within the cell. Both compounds provide reducing equivalents for critical cellular functions and are important for intracellular antioxidant protection. We will first test the role of vitamin C and GSH in EDNO action and production using unstressed HAECs. We will determine the dependence of EDNO action on the cellular content and redox state of vitamin C and GSH. once this is established, we will investigate operative mechanisms such as eNOS activity, cofactor availability, eNOS, auto-inactivation and NO interaction with superoxide. The role of vitamin C and GSH will also be tested in HAECs exposed to physiologically relevant sources of oxidative stress such as superoxide, peroxides, or oxidized LDL. Finally, the roles of vitamin C and GSH on EDNO action in vivo will be tested in guinea pig models. Guinea pigs will be rendered marginally vitamin C and/or GSH deficient through dietary of pharmacologic means and the implications for EDNO action determined using bioassays and related to markers of oxidative stress. Cholesterol -feeding will be employed as a physiologic source of oxidative stress and the superimposed effects of vitamin C and GSH explored in this model as well. These studies should provide additional insight into the role(s) of vitamin C and GSH in maintaining vascular homeostasis and may suggest treatment strategies for patients with atherosclerotic vascular disease.